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Title
Evaluation of Pro-Inflammatory Cytokines Expression in Mouse Splenocytes After
Incubation with Biofield Treated Herbomineral Formulation: Effect of Biofield Energy
Healing Treatment - The Trivedi Effect®
Permalink
https://escholarship.org/uc/item/02j2615j
Journal
American Journal of Biomedical and Life Sciences, 4(6)
ISSN
2330-880X
Authors
Trivedi, Mahendra Kumar
Branton, Alice
Trivedi, Dahryn
et al.
Publication Date
2016-12-08
DOI
10.11648/j.ajbls.20160406.12
Peer reviewed
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University of California
American Journal of Biomedical and Life Sciences
2016; 4(6): 87-97
http://www.sciencepublishinggroup.com/j/ajbls
doi: 10.11648/j.ajbls.20160406.12
ISSN: 2330-8818 (Print); ISSN: 2330-880X (Online)
Evaluation of Pro-Inflammatory Cytokines Expression in
Mouse Splenocytes After Incubation with Biofield Treated
Herbomineral Formulation: Effect of Biofield Energy
®
Healing Treatment - The Trivedi Effect
Mahendra Kumar Trivedi1, Alice Branton1, Dahryn Trivedi1, Gopal Nayak1, Alan Joseph Balmer1,
Dimitrius Anagnos1, Janice Patricia Kinney1, Joni Marie Holling1, Joy Angevin Balmer1,
Lauree Ann Duprey-Reed1, Vaibhav Rajan Parulkar1, Mayank Gangwar2, Snehasis Jana2, *
1Trivedi Global, Inc., Henderson, Nevada, USA
2Trivedi Science Research Laboratory Pvt. Ltd., Bhopal, Madhya Pradesh, India
Email address:
[email protected] (S. Jana)
*Corresponding author
To cite this article:
Mahendra Kumar Trivedi, Alice Branton, Dahryn Trivedi, Gopal Nayak, Alan Joseph Balmer, Dimitrius Anagnos, Janice Patricia Kinney,
Joni Marie Holling, Joy Angevin Balmer, Lauree Ann Duprey-Reed, Vaibhav Rajan Parulkar, Mayank Gangwar, Snehasis Jana. Evaluation of
Pro-Inflammatory Cytokines Expression in Mouse Splenocytes After Incubation with Biofield Treated Herbomineral Formulation: Effect of
Biofield Energy Healing Treatment - The Trivedi Effect®. American Journal of Biomedical and Life Sciences. Vol. 4, No. 6, 2016, pp. 87-97.
doi: 10.11648/j.ajbls.20160406.12
Received: November 3, 2016; Accepted: November 17, 2016; Published: December 8, 2016
Abstract: The use of herbomineral formulations in the healthcare sector has increased due to their high safety and better
therapeutic action. A new proprietary herbomineral formulation was formulated with a mixture of the herbal root extract of
ashwagandha and three minerals viz. zinc chloride, magnesium gluconate, and sodium selenate. The aim of the study was to
evaluate the immunomodulatory potential of Biofield Energy Healing (The Trivedi Effect®) on the formulation when applied to
splenocyte cells isolated from mice spleen. The formulation was divided into two parts; one was the control without any
Biofield Energy Treatment, while the other part was defined as the Biofield Energy Treated sample, which received Biofield
Energy Healing Treatment remotely by seven renowned Biofield Energy Healers. The test formulation was evaluated to find
the expression of pro-inflammatory cytokines such as TNF-α, MIP-1α, and IL-1β along with non-cytotoxic concentrations by
MTT assay. The splenocytes were given the Biofield Energy Treated and untreated sample at concentrations range
(0.00001053 to 10.53 µg/mL) for 48 hours and was reported with safe concentration up to 1.053 µg/mL with percentage
viability range from 76.7% to 109.2% in both samples. Biofield Energy Healing significantly enhanced the cell viability as
compared with the untreated formulation. The expression of TNF-α was significantly inhibited in the Biofield Treated
formulation at 0.01053, 0.1053, and 1.053 µg/mL by 1.77%, 1.93%, and 3.55%, respectively compared with the untreated
formulation. The rest of the tested concentrations of the Biofield Treated formulation showed an increase in TNF-α expression
at 0.00001053, 0.0001053, and 0.001053 µg/mL by 7.26%, 8.50%, and 8.50%, respectively compared to the vehicle control
group. Similarly, the MIP-1α expression was inhibited by the Biofield Energy Treated formulation and showed
immunosuppression activity at 0.01053 µg/mL by 18.47% (p≤0.001) compared to the untreated formulation. MIP-1α
expression was reported as 628.94 ± 13.0 pg/mL in the untreated formulation, while it decreased to 512.74 ± 1.9 pg/mL in the
Biofield Treated formulation at 0.01053 µg/mL. In addition, the IL-1β secretion was also significantly inhibited by the Biofield
Treated formulation at concentrations 0.001053, 0.01053, 0.1053, and 1.053 µg/mL by 72.02%, 50.16%, 30.68%, and 22.11%,
respectively as compared with the untreated formulation. Overall, The Trivedi Effect® significantly down-regulated the pro-
inflammatory cytokines and potentiated the immunosuppressive effect of the treated formulation, which can be better utilized
in organ transplants, anti-aging, stress management, autoimmune disorders, and inflammatory disorders, etc. to modulate the
immune system by improving overall health.
88 Mahendra Kumar Trivedi et al.: Evaluation of Pro-Inflammatory Cytokines Expression in Mouse Splenocytes After Incubation
with Biofield Treated Herbomineral Formulation: Effect of Biofield Energy Healing Treatment - The Trivedi Effect®
Keywords: Biofield Energy Healing Treatment, Biofield Energy Healers, The Trivedi Effect®, LPS,
Pro-Inflammatory Cytokines, TNF-α, MIP-1α, IL-1β
National Center for Complementary and Integrative Health
1. Introduction
(NCCIH). Human Biofield Energy has subtle energy that has
the capacity to work in an effective manner [14]. Reports
The healing properties of plant extracts have been
show that Complementary and Alternative Medicine (CAM)
recognized and utilized worldwide since ancient times. Plant
therapies have been practiced worldwide with reported
products and their extracts are used in both allopathic health
clinical benefits in different health disease profiles [15]. This
care as well as complementary and alternative health care in
energy can be harnessed and transmitted by individuals into
order to improve overall health and the immune system [1,
living and non-living things via the process of Biofield
2]. However, much attention has been focused on discovering
Energy Healing. Biofield Energy Treatment (The Trivedi
herbal products with immunomodulatory activity along with
Effect®) has been extensively studied with significant
low toxicity and better bioavailability [3]. Many scientific
outcomes in many scientific fields such as cancer research
studies have identified the immunomodulatory properties of
[16, 17], altered antimicrobial sensitivity of pathogenic
medicinal plants, which can be further potentiated with the
microbes in microbiology [18-21], genetics [22, 23], altered
addition of some minerals that regulate the immune cells.
physical and chemical properties of pharmaceutical
These types of formulations are commonly defined as
compounds [24-27], improved overall growth and yield of
herbomineral formulations and are the major target for
plants in agricultural science [28-31], and in changing the
pharmaceutical companies as phytopharmaceutical products
structure of the atom in relation to various metals, ceramics,
or as dietary supplements. Based on the literature, a new
polymers and chemicals in materials science [32-35].
proprietary herbomineral formulation was formulated with a
The authors of this study want to evaluate the impact of
combination of the herb ashwagandha root extract and three
Biofield Energy Treatment (The Trivedi Effect®) on the given
minerals viz. zinc, magnesium, and selenium. All the
herbomineral formulation, which might improve the
ingredients of the formulation in this present study possess
immunomodulatory function in in vitro cellular models on
important activities such as immune modulating properties,
mice splenocyte cells.
anti-inflammatory, antioxidant, anti-infective, and anti-viral
[4-7]. Withania somnifera (ashwagandha) biological activity
2. Materials and Methods
is mainly reported due to the presence of withanolides, and it
is used as complementary medicine in alternative therapies
2.1. Chemicals and Reagents
[8, 9]. Apart from its common attributes such as antibacterial,
immunomodulatory and antitumor effects, many clinical and
Lipopolysaccharide (LPS), 3-(4, 5-diamethyl-2-thiazolyl)
preclinical data have been available with respect to its
2, 5 diphenyl-2 H-tetrazolium) (MTT), Roswell Park
immunomodulatory impact [4, 10]. The importance of
Memorial Institute (RPMI-1640), L-glutamine, penicillin,
minerals such as selenium, zinc, and magnesium is to
streptomycin, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
modulate the immune system because their synergistic
acid (HEPES), 2- mercaptoethanol, concanavalin A (Con-A),
impact has been well-defined [5].
rapamycin, NaHCO , and EDTA were purchased from Sigma
3
Scientific research has documented that in the presence of
Chemical Corp. (St. Louis, MO), a subsidiary of Sigma-
minerals, herbal medicines have been found to exhibit a high
Aldrich Corporation. ELISA (enzyme-link immunosorbent
level of phagocytic index and improved antibody titre [11].
assay) assay kits for all cytokines tumor necrosis factor alpha
These formulations can be used for better therapeutic effect in
(TNF-α), macrophage inflammatory protein-1α (MIP-1α),
immune compromised patients affected with cardiovascular
and interleukin-1 beta (IL-1β) were purchased from R&D
diseases, age and stress related diseases, cancer, and
Systems, USA. Fetal bovine serum (FBS) was purchased
autoimmune disorders. Along with herbomineral formulations,
from GIBCO, USA. All other chemicals used were of
the Biofield Energy Healers in this study have used energy
analytical grade available in India. Ashwagandha (Withania
medicine (Biofield Energy Healing Treatment) as a
somnifera) root extract powder (≥ 5% of total withanolides)
complementary and alternative approach to study the impact of
was procured from Sanat Products Ltd., India. Zinc chloride
Biofield Treatment on the herbomineral formulation for its
and magnesium (II) gluconate hydrate were procured from
immunomodulatory potential with respect to the pro-
Tokyo Chemical Industry Co., Ltd. (TCI), Japan. Sodium
inflammatory cytokines in splenocyte cells isolated from mice.
selenate was procured from Alfa Aesar, USA.
In recent years, several scientific reports along with
clinical trials have shown the useful effects of Biofield 2.2. Test Formulation and Reference Standard
Energy Treatment, which has shown enhanced immune
The test formulation contained a combination of four
function in cases of cervical cancer patients with therapeutic
ingredients: ashwagandha root powder extract, zinc chloride,
touch [12], massage therapy [13], etc. Complementary and
sodium selenate, and magnesium gluconate. LPS was used as
Alternative Medicine (CAM) are recognized therapies by The
an inflammatory stimulant, while Con-A and rapamycin were
American Journal of Biomedical and Life Sciences 2016; 4(6): 87-97 89
used as a reference standard (positive control) for 2.6. Isolation of Murine Splenocytes
immunostimulatory and immunosuppressive action
C57BL/6 male mice were sacrificed and the spleens were
respectively in splenocytes assay.
aseptically removed and grounded by passing through a
2.3. Experimental Animal sterile plastic strainer under aseptic conditions. After the cells
were centrifuged twice at 1000 g for 5 minutes, erythrocytes
C57BL/6 male mice (8 weeks old, 22 gm body weight)
were lysed by a lysis buffer (0.15 M NH Cl, 0.01 M
4
were purchased from Vivo Bio Tech Ltd., Hyderabad, India
NaHCO , and 0.1 mM EDTA, pH 7.4) and then the cell
3
and acclimatized for one week prior to the experiments.
pellets were washed twice with the RPMI-1640 medium.
Laboratory rodent diet and drinking tap water were provided
Further, the cells were resuspended in the complete RPMI-
ad libitum and were maintained under controlled conditions
1640 medium (RPMI 1640 medium plus 10% fetal bovine
with a temperature of 22 ± 3°C, humidity of 30% to 70% and
serum, 2 mM glutamine, 100 IU/mL of penicillin and
a 12-hour light/12-hour dark cycle. All the procedures were
streptomycin, 15 mM HEPES and 50 mM 2-
in strict accordance with the Guide for the Care and Use of
mercaptoethanol). The cell counts were performed using a
Laboratory Animals published by the US National Institutes
hemocytometer and cell viability was determined using the
of Health (NIH). The approval of the Institutional Animal
trypan-blue dye exclusion technique with the results showing
Ethics Committee (IAEC) was obtained prior to carrying out
≥95% of viable cells. The cells were cultured in 96-well
the animal experiment. tissue culture plates with 0.2 x 106 cells per well. They were
incubated at 37°C in a humidified atmosphere of 5% CO for
2.4. Biofield Energy Healing Strategies 2
the indicated period [36].
One part of the test formulation did not receive any sort of
2.7. Cell Culture and Test Formulation Treatment
treatment and was defined as the control group, while
Biofield Energy Treatment was given to the herbomineral Splenocyte (0.2 x 106 cells per well) cells were grown in
formulation defined as the treated group. The Biofield
96-well culture plates using a RPMI-1640 medium
Energy Treatment was administered for 5 minutes by the
supplemented with 10% FBS, 100 units/mL of penicillin, and
seven Biofield Energy Healers (The Trivedi Effect®), six of
100 µg/mL of streptomycin. LPS (50 ng/mL) induced
which were remotely located in the U. S. A. and one of splenocyte cells cultures were grown for 48 hours at 37oC in
which was remotely located in Canada, while the test
a humidified CO incubator (5% CO ). The effect of
2 2
formulation was located in Dabur Research Foundation near
cytotoxicity of the formulation was tested by treating cells
New Delhi in Ghaziabad, India, under standard laboratory
with different concentrations of the test formulation in
conditions. This treatment was provided through the Biofield
RPMI-1640 medium. The various concentrations of the test
Energy Healers’ unique Energy Transmission process (The
formulation were used i.e. 0.00001053 µg/mL to 10.53
Trivedi Effect®) remotely to the test formulation. Further, the
µg/mL in the presence of inflammatory stimulus (LPS) for
control group was treated by a sham healer for comparative
cell viability assay. The respective vehicle controls (DMSO)
purposes. The sham healer does not have any knowledge
were kept in the assay for comparison.
about the Biofield Energy Treatment. After that, the Biofield
Energy Treated and untreated samples were kept in similar 2.8. Cytotoxicity by MTT Assay
sealed conditions and used for the in vitro study on
The effect of the Biofield Treated and untreated
splenocyte cells for cytokines estimation.
formulations at the concentration range of 0.00001053
2.5. Experimental Design µg/mL to 10.53 µg/mL were tested for cell viability using 3-
(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide
The experimental study was divided into 7 groups. Group 1
(MTT) assay. The number of viable cells was determined by
comprised of the splenocyte cells without LPS and was
the ability of mitochondria to convert MTT to formazan dye.
denoted as the negative control. Group 2 served as a
Splenocyte cells were cultured overnight in 96-well plates, at
stimulant group that includes cells with LPS. Group 3 a density of 0.2 x 106 cells per well. After treatment with the
included the splenocyte cells with LPS along with vehicle
test formulation and incubation period, the medium was
(0.005% DMSO) denoted as the vehicle control. Groups 4
removed. 20 µL of 5 mg/mL MTT was then added to each
and 5 were defined as the positive control, which includes
well and incubated for 3 hours further at 37ºC in a humidified
cells with Con-A (0.5 μg/mL) and rapamycin (1 nm and 10
5% CO atmosphere. The cells were centrifuged and
2
nm), respectively. Groups 6 and 7 were denoted as the test
supernatants were removed. The cell pellet in each well was
item groups that included splenocyte cells with LPS along
resuspended in 150 µL of DMSO to dissolve formazan
with untreated and Biofield Treated formulations,
crystals. The optical density of each well was read at 540 nm
respectively, at concentration 0.00001053 to 10.53 µg/mL.
using BioTek Reader (SIAFRT/Synergy HT multimode
After 48 hours of incubation, supernatants were analyzed for
reader, US).
the secreted levels of TNF-α, MIP-1α, and IL-1β using
The effect of the formulation on cell viability of splenocyte
ELISA as per the manufacturer’s instructions. Concentrations
cells was determined as per equation (1):
were determined in triplicate wells of each sample.
90 Mahendra Kumar Trivedi et al.: Evaluation of Pro-Inflammatory Cytokines Expression in Mouse Splenocytes After Incubation
with Biofield Treated Herbomineral Formulation: Effect of Biofield Energy Healing Treatment - The Trivedi Effect®
% Cell viability(cid:12)100(cid:15)% cytotoxicity (1) room temperature and the plate was incubated for 45 minutes
at room temperature with gentle shaking. The plates were
Where; % cytotoxicity = [(O. D. of control cells – O. D. of
again washed 3 times and then 100 µL of horseradish per-
cells treated with the test formulation)/O. D. of control
oxidase (HRP)–streptavidin conjugate solution was added
cells]*100.
and the plate was incubated for 45 minutes at room
The concentration that resulted in >75% viability was
temperature with gentle shaking. Next, the plate wells were
selected for subsequent cytokine estimation.
washed 3 times as previous and 100 µL of 3,3,5,5'-
tetramethylbenzidine (TMB) one-step substrate reagent was
2.9. Determination of Cytokines (TNF-α and IL-1β) and
added, followed by a 30-minute incubation at room
Chemokine (MIP-1α) Using ELISA
temperature in the dark. Further, 50 µL of 0.2 mol/L
The in-vitro activity of the Biofield Treated and untreated sulphuric acid was added to each well to stop the reaction
test formulations were estimated on the mice splenocytes for and the plates were read for absorbance at 450 nm using a
the production of TNF-α, MIP-1α, and IL-1β using enzyme- BioTek Reader (SIAFRT/Synergy HT multimode reader).
linked immunosorbent assay (ELISA). The ELISA plates Standards were run in parallel to the samples, and the
were coated with an antibody in a coating buffer at the concentrations were determined in triplicate for each sample
recommended concentration and kept overnight at 4ºC. After [37].
washing with PBS-T (PBS with 0.05% Tween 20), the plates
2.10. Statistical Analysis
were blocked with assay diluent for at least 2 hours at room
temperature. A total of 100 µL culture supernatant from
Data were expressed as mean ± SEM and were subjected
different experimental samples and standards were incubated
to one-way analysis of variance (ANOVA) followed by
overnight at 4ºC and, after three washes, biotinylated anti-
Dunnett’s method and Student’s t-test for two groups
mice cytokine (TNF-α, MIP-1α, and IL-1β) antibodies at the
comparison. Statistical significance was considered at
recommended concentrations were incubated for 1 hour at
p≤0.05.
Figure 1. MTT assay in the splenocyte cells after the 48-hours of treatment with different test formulation concentrations in the presence of 0.5 µg/mL LPS.
The absorbance of the MTT formazan was determined at 540 nm in an ELISA reader. Cell viability was defined as the absorbance ratio (expressed as a
percentage) of the test formulation treated cells relative to the untreated vehicle control group.
concentrations 1 and 10 nm, respectively. The increased cell
3. Results viability with respect to vehicle control might be due to
proliferation in cell culture. Con-A and rapamycin showed
3.1. MTT Assay on Splenocyte Cells immunostimulatory and immunosuppressive action,
respectively, as used as positive control in the experiment.
The effect of the Biofield Treated and untreated test
Concentration range of 0.00001053 to 10.53 µg/mL was
formulations on the proliferation of mice splenocyte cells
selected for comparison of cell viability after adding the
was examined after 48 hours through MTT cell viability
Biofield Treated and untreated test formulations to the
assay. The effect of the test formulation on the viability of the
splenocyte cells. The test formulation was found safe at
splenocytes is shown in Figure 1. The results showed the %
concentration up to 1.053 µg/mL with percentage viability
cell viability was altered after Biofield Treatment in the
range from 76.7% and 109.2%. The cell viability was greatly
tested concentration of the test formulation. The untreated,
decreased at the concentration of 10.53 µg/mL in both the
LPS, and Con-A group showed 100%, 171.7%, and 201.9%
Biofield Treated (20.9%) and the untreated formulations
cell viability, respectively. The vehicle control group reported
(33.9%), so this concentration was not selected for the
with 100% and the rapamycin group as 98.9% and 89.9% at
American Journal of Biomedical and Life Sciences 2016; 4(6): 87-97 91
estimation of cytokines. However, at three tested formulations on TNF-α secretions in splenocyte cells are
concentrations the Biofield Treated test formulation showed represented in Figure 2. Results suggest that both the
increased cell viability, i.e. at 0.0001053, 0.01053, and 1.053 untreated and Biofield Treated groups demonstrated
µg/mL, while the rest of the three concentrations showed suppression of TNF-α secretions at different tested
decreased cell viability i.e. at 0.00001053, 0.001053, and concentrations i.e. at 0.00001053 to 1.053 µg/mL. The
0.1053 µg/mL with respect to the vehicle control group. The negative control (untreated cells), LPS, Con-A, and vehicle
percentage of increased cell viability in the Biofield Treated control group showed TNF-α values as 87.60, 504.65,
test formulation was 1.00%, 2.89%, and 0.29% at 0.0001053, 271.32, and 501.55 pg/mL, respectively. However, the
0.001053, and 1.053 µg/mL respectively as compared with untreated test formulation demonstrated suppression of
the untreated test formulation. Similarly, the concentrations TNF-α from LPS stimulated levels at five tested
0.0001053, 0.01053, and 1.053 µg/mL showed increased cell formulation concentrations out of six i.e. at 0.0001053,
viability as 0.9%, 9.2%, and 5.5%, respectively as compared 0.001053, 0.01053, 0.1053, and 1.053 µg/mL by 12.51%,
with the vehicle control group. The percentage of decreased 7.50%, 6.49%, 5.80%, and 16.46%, respectively as
cell viability reported at 0.00001053, 0.01053, and 0.1053 compared with the vehicle control. The maximum
µg/mL was 22.36%, 2.58%, and 14.54%, respectively in decreased value of TNF-α of the untreated test formulation
comparison to the untreated test formulation. Overall, the was reported at 1.053 µg/mL i.e. 418.99 pg/mL. At three
results showed that the cell viability was significantly altered tested concentrations, the Biofield Treated test formulation
after the Biofield Energy Treatment. showed an inhibition of TNF-α i.e. at 0.01053, 0.1053, and
1.053 µg/mL by 1.77%, 1.93%, and 3.55%, respectively as
3.2. Effect of the Test Formulation on the Expression of
compared to the vehicle control group. The maximum
Pro-Inflammatory Cytokines (TNF-α and IL-1β) and
inhibition of TNF-α in the Biofield Energy Treated test
Chemokine (MIP-1α) in the Splenocyte Cells
formulation was reported (483.72 pg/mL) at the test
formulation concentration 1.053 µg/mL as compared to the
The effect of the Biofield Treated herbomineral
vehicle control. On the other hand, the Biofield Energy
formulation was observed on pro-inflammatory cytokines
Treated test formulation demonstrated an increase in TNF-α
TNF-α, MIP-1α, and IL-1β. All play a major role in
at three tested formulation concentrations i.e. 0.00001053,
inflammation, immune modulation, and lymphocyte
0.0001053, and 0.001053 µg/mL by 7.26%, 8.50%, and
activation, so it might be expected that the herbomineral
8.50%, respectively as compared to the vehicle control
formulations can modulate the expression and activation of
group. Overall, it can be suggested that the test formulation
cytokines. Therefore, the expression of TNF-α, MIP-1α and
has immunosuppressive effects by inhibiting the
IL-1β at six concentrations was examined in the spleen cells.
concentration of TNF-α as compared with the vehicle
The effect of the test formulation on pro-inflammatory
control, while the Biofield Energy Treatment has increased
cytokines was estimated by incubating the formulation with
the concentration of TNF-α in all the concentrations as
various concentrations of the treated and untreated test
compared with the untreated formulation. The Biofield
formulations for 48 hours using ELISA assay.
Energy Treatment showed a significant effect on altering
3.2.1. Assessment of TNF-α Expression the level of TNF-α as compared to the untreated test
The effect of the Biofield Treated and untreated test formulation.
Figure 2. Concentration-dependent effect on TNF-α by the Biofield Treated and untreated test formulations. For each concentration treatment, the level of
TNF-α release was measured after receiving 48-hours of treatment. All values are represented (in pg/mL) as mean ± SEM.
92 Mahendra Kumar Trivedi et al.: Evaluation of Pro-Inflammatory Cytokines Expression in Mouse Splenocytes After Incubation
with Biofield Treated Herbomineral Formulation: Effect of Biofield Energy Healing Treatment - The Trivedi Effect®
3.2.2. Estimation of MIP-1α Expression compared to the vehicle control group. The Biofield Energy
The effect of the Biofield Energy Treated and untreated Treatment further enhanced the immunosuppressive property
test formulations on MIP-1α secretion is shown in Figure 3. of the test formulation by 18.47%. The enhanced activity of
The figure demonstrates that the Biofield Treated and MIP-1α expression was reported at concentration 0.01053
untreated test formulations inhibit the expression of MIP-1α µg/mL i.e. 512.74 ± 1.9 pg/mL in the Biofield Treated test
as compared with the vehicle control group. However, the formulation and 628.94 ± 13.0 pg/mL in the untreated
comparative effect of the Biofield Energy Treated and formulation. It showed significant decreased level of MIP-1α
untreated test formulations on MIP-1α secretion in by 18.47% (p≤0.001) as compared to the untreated
splenocyte cells showed significant alterations at all the formulation. The rest of the other Biofield Treated test
tested concentrations. The untreated cells, LPS, Con-A, and formulation concentrations showed an increased level of
vehicle control group showed values of MIP-1α as 42.8 ± 14, MIP-1α as compared with the untreated test formulation.
598.6 ± 11.2, 285.5 ± 10.3, and 768.0 ± 10.2 pg/mL, However, the Biofield Energy Treated test formulation group
respectively. The untreated test formulation showed reported an inhibition of MIP-1α secretion at 0.00001053,
significant inhibition of MIP-1α secretion at all the tested 0.0001053, 0.001053, 0.01053, 0.1053, and 1.053 µg/mL by
concentrations i.e. at 0.00001053, 0.0001053, 0.001053, 25.41%, 25.71%, 35.55%, 33.24%, 19.18%, and 27.78%,
0.01053, 0.1053, and 1.053 µg/mL by 24.27%, 41.30%, respectively as compared with the vehicle control group.
45.72%, 18.11%, 19.18%, and 36.15%, respectively as
Figure 3. Concentration-dependent inhibition of LPS mediated production of MIP-1α by the Biofield Treated and untreated test formulations. For each
concentration treatment, the level of MIP-1α release was measured after receiving 48-hours of treatment. The values are represented in pg/mL as mean ± SEM
(***p≤0.001, **p≤0.01, and *p≤0.05, as compared with the untreated test formulation).
control group. The Biofield Treatment showed further
3.2.3. Estimation of IL-1β Expression
significant improvement in the immunosuppressive property
The effect of the Biofield Treated and untreated test
of the test formulation at all the concentrations, except at
formulations on IL-1β expression is shown in Figure 4. The
0.00001053 µg/mL, as compared to the untreated
figure demonstrates the inhibition of IL-1β after treatment
formulation. The significant inhibition of IL-1β secretion
with the Biofield Energy Treated and untreated test
after the Biofield Treatment was reported at the
formulations as compared with the vehicle control group.
concentrations viz. 0.001053, 0.01053, 0.1053, and 1.053
However, the comparative effect of the treated and untreated
µg/mL by 72.02%, 50.16%, 30.68%, and 22.11%,
test formulations on IL-1β secretion in splenocyte cells
respectively as compared with the untreated formulation. The
showed significant inhibition at 5 tested concentrations out of
maximum IL-1β secretion inhibition was reported in the
6. The untreated cells, LPS, Con-A, and vehicle control
Biofield Treated test formulation at 0.001053 µg/mL. While
group showed values of IL-1β as 7.67 ± 1.9, 78.86 ± 5.8,
60.17 ± 7.7 was reported in the untreated test formulation,
19.48 ± 4.5, and 54.90 ± 3.4 pg/mL, respectively. The
the Biofield Treated sample reported 16.84 ± 1.8 pg/mL
untreated test formulation showed a significant inhibition of
(p≤0.01). However, the Biofield Treated group reported an
IL-1β secretion at all the tested concentrations i.e. at
inhibition of IL-1β secretion at 0.00001053, 0.0001053,
0.00001053, 0.0001053, 0.001053, 0.01053, 0.1053, and
0.001053, 0.01053, 0.1053, and 1.053 µg/mL by 35.36%,
1.053 µg/mL by 77.21%, 40.13%, 31.73%, 36.62%, 43.24%,
43.24%, 80.89%, 68.41%, 60.65%, and 55.42%, respectively
and 42.78%, respectively as compared with the vehicle
as compared with the vehicle control group.
American Journal of Biomedical and Life Sciences 2016; 4(6): 87-97 93
Figure 4. Concentration-dependent inhibition of LPS mediated production of IL-1β by the test formulations. For each concentration treatment, the level of IL-
1β release was measured in cell supernatant after receiving 48-hours of treatment. All values are represented in pg/mL as mean ± SEM (**p≤0.01, as
compared with the untreated test formulation).
could affect the course of immune response and the whole
4. Discussion
network of immune regulation. The individual components,
such as ashwagandha, have been reported to inhibit the NF-
Traditional, alternative and complementary medicines and
κB and AP-1 transcription factors in human peripheral blood
therapies play a major role worldwide with respect to the
and synovial fluid mononuclear cells [42]. With respect to the
current human wellness and traditional healthcare sectors,
minerals present in the formulation, the deficiency of zinc
including overall healthcare management, longevity, anti-
directly influenced the cytokines generation such as IL-2, IL-
aging and quality of life. However, more attention is required
6, IL-1β, and TNF-α, and influenced its generation in a
to understand the use of alternative energy medicine and its
concentration depended manner. It was reported that zinc
utilization in current healthcare systems. The scope of
also alters the cytokines generation by inhibiting the
traditional medicine has only been improved in developing
activation of NF-κB [43].
countries [38], but the use of Biofield Energy as a
Magnesium also plays an important role in the activation
Complementary and Alternative Medicine (CAM) approach
of NF-κB, and affects the generation of cytokines, and would
still requires implementation in the practice by many
be effective in the risk situations associated with
scientific research. The herbal medicine industry is
inflammation or its related diseases pathogenesis [44].
increasing worldwide more than 10% annually, and it may
However, selenium from the diet mostly incorporates into
reach up to $5 trillion by the year 2050 [39]. Many
selenoproteins, and plays an important role in inflammation
herbomineral products have shown significant effect in
and for initiating immunity. Selenium influences different
curing many diseases with minimal adverse effects. One
leukocytes effector functions such as cytokines secretion,
widely used example of an herbomineral formulation is the
migration, adherence, and phagocytosis. It plays an important
Naga bhasma in the Indian system of medicines, which is
role in cytokine production using calcium flux and oxidative
used for various disorders such as diabetes, indigestion,
pathway [45-47].
inflammation of the intestine, osteomalacia, blood disorders,
Study results suggest that the Biofield Treated test
infertility, regenerate germinal epithelium of the testes, as a
formulation showed an anti-inflammatory effect and
potent revitalizer, and many more uses within our bodies
[40]. The Trivedi Effect®-Biofield Energy Healing (TEBEH) suppressed the level of tested cytokines as compared with the
vehicle control group. However, the Biofield Energy Treated
is a new proprietary method that can be used to enhance the
formulation further potentiated the effect as compared with
impact of herbomineral formulations, which can be further
the untreated test formulation, which may better prevent the
used to modulate the immune function with long-term
progression of acute or chronic infection, because
effectiveness and minimal toxicity at a lower cost, which
inflammation is regarded as the first response that releases
might improve overall health and quality of life.
cytokines. Thymus and spleen cell proliferation are
The individual ingredients of the herbomineral
considered as vital parameters to maintain immune
formulation, including ashwagandha and zinc chloride, have
homeostasis [48]. Inhibition of cytokines such as TNF-α, IL-
already been reported in many scientific reports to modulate
1, and IL-6, and eicosanoid such as PGE2 from any
the inflammatory response using cytokines estimation [4-7].
formulation can be considered to have anti-inflammatory
Lymphocyte proliferation and the activation of NK cells are
properties, and the Biofield Energy Treated test formulation
cytokine dependent [41], and its up and down regulation
94 Mahendra Kumar Trivedi et al.: Evaluation of Pro-Inflammatory Cytokines Expression in Mouse Splenocytes After Incubation
with Biofield Treated Herbomineral Formulation: Effect of Biofield Energy Healing Treatment - The Trivedi Effect®
can be better utilized as an anti-inflammatory agent in stress, and asthma [54].
comparison to the untreated test formulation. Biofield Energy Healing has been succesfully reported in
Cells viability assay (MTT) suggest that the Biofield the case of cancer cell lines with respect to the inhibition of
Treated and untreated test formulations’ concentration was cytokine expression such as IL-1α and IL-1β levels [55].
found safe with respect to the in vitro viability of splenocytes Complementary and alternative medicine (energy therapy)
until concentration 1.053 µg/mL, while the viability has been reported with various benefits as compared with the
percentage was increased after the Biofield Energy conventional treatment approach. The National Center for
Treatment. The MTT assay evaluates the metabolic activity Complementary and Integrative Health (NCCIH) under the
by measuring the activity of succinate dehydrogenase, a National Institutes of Health (NIH) have recognized the
mitochondrial enzyme. This test is widely used in the in vitro siginificant outcomes of energy medicine [56].
evaluation of the cell toxicity of any test drug. MTT assay is
regarded as more rapid, less costly, less time consuming, and 5. Conclusions
as a non-radioactive method to show the cell proliferation
results on the basis of cell growth and metabolic activity The in vitro results of the Biofield Energy Treated and
[49]. Further, the level of TNF-α was inhibited at three tested untreated test formulations showed significant modulatory
higher concentrations i.e. 0.01053, 0.1053, and 1.053 µg/mL effects on splenocyte cells isolated from mice with respect to
by 1.77%, 1.93%, and 3.55%, respectively by the Biofield the suppression of tested cytokines. All the tested cytokines
Treated test formulation. Thus, it can be concluded that the (TNF-α, MIP-1α, and IL-1β) were reported with
Biofield Energy Treatment potentiated the inhibition and immunosuppressive activity with respect to the vehicle
showed immunosuppressive activity at higher concentrations. control group, while the Biofield Energy Treatment further
TNF-α plays a major role in immune disorders, and is the potentiated the immunomodulation as compared with the
controlling factor for many diseases [50]. However, the level untreated test formulation. The results of in vitro splenocyte
of MIP-1α was inhibited at all of the Biofield Treated test cells’ viability assay suggest that most of the tested
formulation concentrations with respect to the vehicle concentration of the Biofield Treated and untreated test
control, while showing significant inhibition at 0.01053 formulations showed improved cell viability, except at the
µg/mL i.e. 18.74% with respect to the untreated test higher concentration of 10.53 µg/mL. The cell viability after
formulation. Reduction of MIP-1α levels might be useful in the Biofield Energy Treatment significantly improved with
decreasing the inflammatory responses [51]. the Biofield Treated test formulation at concentrations
Similarly, the expression of IL-1β was further significantly 0.0001053, 0.001053, and 1.053 µg/mL with respect to the
potentiated after the Biofield Treatment on the test untreated test formulation. The results of the cytokines
formulation at all concentrations, except lower concentration estimation revealed that the Biofield Treated group
values i.e. 0.00001053 µg/mL. The percentage of inhibition suppressed the TNF-α, MIP-1α and IL-1β. TNF-α levels,
ranged from 22.11% to 72.02% in the Biofield Treated test which were significantly inhibited at three tested
formulation as compared with the untreated test formulation. concentrations of 0.01053, 0.1053, and 1.053 µg/mL by
Immunological and inflammatory functions of IL-1β in 1.77%, 1.93%, and 3.55%, respectively in the Biofield
controlling the immune response during infections are well Treated test formulation as compared with the untreated test
defined [52, 53]. Overall, the inhibitory effect might be the formulation. In the case of MIP-1α, both the untreated and
result of specific inhibition of NF-κB, a transcription factor Biofield Treated test formulation showed significant
involved in the activation of many inflammatory mediator suppression, while the Biofield Energy Treatment enhanced
genes. The results also reflect that the Biofield Treated test the immunosuppressive activity at the concentration 0.01053
formulation showed immunosuppressive activity in LPS µg/mL by 18.47% (p≤0.001) as compared to the untreated
stimulated splenocyte cells on pro-inflammatory cytokines formulation. The values of MIP-1α reported in the case of the
(TNF-α, MIP-1α, and IL-1β) secretion, which can be untreated formulation was 628.94 ± 13.0 pg/mL, while it was
concluded that the Biofield Treated test formulation has the decreased to 512.74 ± 1.9 pg/mL in the Biofield Energy
potential to act on the innate immune system. LPS was used Treated test formulation. Similarly, significant inhibition
in the study to induce the immune system to depict the (p≤0.001) of IL-1β secretion was reported in the Biofield
disease state. The effect was significant with respect to all Treated test formulation at concentration 0.001053, 0.01053,
cytokines as they are deeply associated with inflammatory 0.1053, and 1.053 µg/mL by 72.02%, 50.16%, 30.68%, and
disorders (such as TNF-α and IL-1β for chronic 22.11%, respectively as compared with the untreated
inflammation). In the case of many autoimmune disorders, formulation.
such as rheumatoid arthritis, steroids (prednisolone and On the basis of the above estimation, it can be concluded
dexamethasone) are preferred to suppress the cytokines that after receiving the Biofield Energy Healing Treatment
production. However, this study suggests that a Biofield (The Trivedi Effect®), administered remotely by the seven
Treated formulation would be a better choice of treatment Biofield Energy Healers to the new herbomineral formulation
and has no toxicity. Besides, it can also be preferred for the significantly inhibited the activity of pro-inflammatory
inhibition of T and B lymphocytes, which could help in cytokines that might prevent the over-activation of the
immune-mediated disorders such as autoimmune disorders, immune system. The Biofield Energy Treated test
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during chemoradiation using a novel integrative approach.
LPS: Lipopolysaccharide; DMSO: Dimethyl sulfoxide; Brain Behav Immun 24: 1231-1240.
FBS: Fetal bovine serum; MTT: 3-(4,5-Dimethylthiazol-2-
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NCCAM: National Center for Complementary and
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Description:To cite this article: Mahendra Kumar Trivedi, Alice Branton, Dahryn Trivedi, Gopal Nayak, Alan Joseph Balmer, Dimitrius Anagnos, Janice Patricia Kinney,. Joni Marie Holling, Joy Angevin Balmer, Lauree Ann Duprey-Reed, Vaibhav Rajan Parulkar, Mayank Gangwar, Snehasis Jana. Evaluation of.
